It is known in this field that a full length cottontail rabbit papillomavirus (CRPV) DNA genome of the Washington B strain of the virus can be injected into rabbit skin as an episome by several methods (See, Brandsma et al, Proc. Natl. Acad. Sci., U.S.A., Vol. 88, pages 4816-4820 (June 1991)). In the Brandsma publication, inter alia, the authors discuss, as one of the several methods of inoculating with the episome, the use of a ped-o-jet (a standard hypodermic jet injector gun) for injecting the entire viral genome of the cottontail rabbit papillomavirus (CRPV) DNA into rabbit skin. Prior to injection of the full length genome Brandsma purifies it either by using standard cesium chloride centrifugation or by using polyethylene glycol precipitation followed by proteinase K digestion. Equivalent purification results were obtained with DNA purified by either method.
The Brandsma publication alludes to the possibility that further research might lead to other forms of DNA that might also be injected using the jet injection technique, but there is no data or descriptional basis to lead one to expect any likelihood of success with injecting DNA if less than a complete genome episome.
The four different methods discussed in Brandsma et al for inoculating rabbit skin involved (a) epithelial scarification with a razor blade followed by smearing on of DNA, (b) scratching the skin with the back of an 18-gauge needle followed by smearing on of DNA, (c) intradermal inoculation and puncture 200 times with a 27-gauge needle, and (d) interdermal inoculation using a PED-O-Jet injector (Stirn Industries, Dayton, N.J.).
The four types of inoculations were performed on four to five pound random-bred New Zealand white female rabbits, anesthetized with ketamine hydrochloride (44 milligrams per kilogram) after clipping their backs free of hair. The inoculant contained 70 micrograms of supercoiled CRPV-p LAI1 DNA (wild type or mutant) per site in 0.1 milliliter of 0.15 M NaCl or 10 mM Tris.HCl/1 mM EDTA.
Inoculation methods (a) and (b) were not very efficient, but the intradermal inoculation and puncture (manual) and the jet injector methods both induced papillomas. The intradermal manual method induced papillomas in 15-25% of the inoculated sites on all the rabbits. The jet-injector caused 23-81% of the inoculated sites to form papillomas.
The Brandsma paper concluded that there was some advantage as to the total number of papillomas produced and a savings of time for the inoculation. However, it was not clear whether the increased rate of producing papillomas from the DNA episomes could also be accounted for possibly of different depths at which the injection was done into the tissue using the manual or the jet method. Further, there is no data for the manual method using a larger diameter (smaller gauge) needle for the injection. The 27 gauge needle used has a very small diameter and could possibly have broken the episome DNA into fragments.
In other words, Brandsma is not clear as to whether the jet injector and the manual injection were done at the same depth in the tissue. Possibly, one of the two methods injected deeper into the tissue or injected over a wider area of tissue. Specifically a different manual injection technique (such as injecting while withdrawing the needle instead of at just one depth) may have been much more successful than the technique used by Brandsma. Thus, the data produced in Brandsma is inconclusive on this point.
Moreover, in Brandsma there is no experimental evidence or data that would provide any reasonable basis for believing that DNA fragments which are not capable of producing an episome would be expressed if they were injected either manually or using the jet injection.
Accordingly, there is a need in this art for an improved method for initiating gene expression for DNA which corresponds to less than a complete genome, i.e., gene fragments, in a host and for improved equipment with which to perform these injections.